Pseudomonas putida is a bacterium which can grow on d-camphor as its sole source of carbon and energy. Under these conditions the organism contains a soluble cytochrome P-450 dependent monooxygenase system which hydroxylates camphor. This monooxygenase system is absent from the bacterium grown on more conventional carbon sources such as glucose or succinate. The membrane bound electron transport system which occurs in this organism is unusual in that the terminal oxygen reductant (cytochrome oxidase) is not a heme a type of cytochrome. The ultimate goal of this project is to obtain an understanding of the interaction between these two oxygen and NADH consuming enzyme systems in this bacterium. The research goals for the current year are: A. Delineate the sequence of electron carriers in membrane electron transport. B. Continue studies of the interaction of putidaredoxin with bacterial cytochrome P-450. These studies will now be extended to include the reaction with oxy-cytochrome P-450 and any intermediates which might occur during oxygen activation. The rapid mixing freeze-quenching device will be used to examine this interaction as it controls the function of cytochrome P-450. C. Establish the optimal conditions for induction of cytochrome P-450 by camphor. The ultimate goal of these studies is to devise conditions for induction so that specifically labeled amino acids can be incorporated into cytochrome P-450 for specific tagging of the protein. BIBLIOGRAPHIC REFERENCES: Griffin, B.W., Peterson, J.A., Werringloer, J., and Estabrook, R.W. (1975) Ann. N.Y. Acad. Sci., 244, 107-131. "Chemistry of Soluble and Membrane-Bound Cytochrome P-450". Rahimtula, A.D., O'Brien, P.J., Hrycay, E.G., Peterson, J.A., and Estabrook, R.W. (1974) Biochem. Biophys. Res. Commun., 60, 695-702. "Possible Higher Valence States of Cytochrome P-450 During Oxidative Reactions".